Bottom Line:
The ability of AGP to bind circulating lipopolysaccharide (LPS) in plasma is believed to help reduce the proinflammatory effect of bacterial lipid A molecules.In order to dissect the contribution of the lipid A, core oligosaccharide and O-antigen polysaccharide components of LPS, the AGP binding affinity of LPS from smooth strains, were compared to lipid A, Kdo2-lipid A, R(a), R(d), and R(e) rough LPS mutants.The SAR analysis enabled by the binding data suggested that, in addition to the important role played by the lipid A and core components of LPS, it is predominately the unique species- and strain-specific carbohydrate structure of the O-antigen polysaccharide that largely determines the binding affinity for AGP.

ABSTRACTThe ability of AGP to bind circulating lipopolysaccharide (LPS) in plasma is believed to help reduce the proinflammatory effect of bacterial lipid A molecules. Here, for the first time we have characterized human AGP binding characteristics of the LPS from a number of pathogenic Gram-negative bacteria: Escherichia coli, Salmonella typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa, and Serratia marcescens. The binding affinity and structure activity relationships (SAR) of the AGP-LPS interactions were characterized by surface plasma resonance (SPR). In order to dissect the contribution of the lipid A, core oligosaccharide and O-antigen polysaccharide components of LPS, the AGP binding affinity of LPS from smooth strains, were compared to lipid A, Kdo2-lipid A, R(a), R(d), and R(e) rough LPS mutants. The SAR analysis enabled by the binding data suggested that, in addition to the important role played by the lipid A and core components of LPS, it is predominately the unique species- and strain-specific carbohydrate structure of the O-antigen polysaccharide that largely determines the binding affinity for AGP. Together, these data are consistent with the role of AGP in the binding and transport of LPS in plasma during acute-phase inflammatory responses to invading Gram-negative bacteria.

Mentions:
The highly variable length of the O-antigen polysaccharide means that LPS from different bacterial strains have different molecular weights which complicates quantitative comparisons of binding affinity. Accordingly, the molar concentration of the LPS samples was determined using the purpald Kdo assay and we standardized the concentrations of LPS in the SPR experiments. The SPR binding measurements were performed using AGP immobilized to a CM5 sensor chip surface and titrated with LPS (see Figure S1 available online at doi:10.1155/2012/475153). In order to investigate the impact of EDTA on binding, the experiments were replicated in HBS-P and HBS-EP buffers (HBS-EP buffer contains 3 mM EDTA). The binding of the LPS samples to AGP was dose-dependent (Figures 3(a) and 3(b)). The binding affinity varied between LPS isolates from different genera and between LPS isolates from different strains (Figure 3). Ranking of LPS binding to AGP was investigated at single concentrations of 40 μM of LPS, which gave a rank order of affinity P. aeruginosa > K. pneumonia > E. coli O127:B8 > E. coli O111:B4 > S. enteric > E. coli EH100 (Ra) ≥ S. enteric TV119 (Ra) > S. enteric SL1181 (Re) > S. marcescens > E. coli F583 (Rd) (Figure 3(b)). The binding of E. coli O111:B4, E. coli O127:B8, E. coli EH100 (Ra), S. enterica TV119 (Ra), and S. enteric SL1181 (Re) was higher in HBS-EP than in HBS-P buffer. Whereas, in the case of K. pneumonia, Pseudomonas aeruginosa, S. enterica, and S. marcescens, the presence of EDTA did not markedly affect the AGP binding levels. Because of its insolubility in the flow buffer, the binding of diphosphoryl lipid A from E. coli was investigated using hybrid SUVs. There was no specific AGP binding detected for diphosphoryl lipid A (data not shown). Hybrid SUV experiments were also performed with fully synthetic Kdo2-lipid A and E. coli F583 (Rd) LPS (Figure 3(d)). There was no specific AGP binding detected for E. coli F583 (Rd) lipid A, while as the concentrations of AGP were higher than 7 μM, a dose-dependent response to Kdo2-lipid A was observed (Figure 3(d)).

Mentions:
The highly variable length of the O-antigen polysaccharide means that LPS from different bacterial strains have different molecular weights which complicates quantitative comparisons of binding affinity. Accordingly, the molar concentration of the LPS samples was determined using the purpald Kdo assay and we standardized the concentrations of LPS in the SPR experiments. The SPR binding measurements were performed using AGP immobilized to a CM5 sensor chip surface and titrated with LPS (see Figure S1 available online at doi:10.1155/2012/475153). In order to investigate the impact of EDTA on binding, the experiments were replicated in HBS-P and HBS-EP buffers (HBS-EP buffer contains 3 mM EDTA). The binding of the LPS samples to AGP was dose-dependent (Figures 3(a) and 3(b)). The binding affinity varied between LPS isolates from different genera and between LPS isolates from different strains (Figure 3). Ranking of LPS binding to AGP was investigated at single concentrations of 40 μM of LPS, which gave a rank order of affinity P. aeruginosa > K. pneumonia > E. coli O127:B8 > E. coli O111:B4 > S. enteric > E. coli EH100 (Ra) ≥ S. enteric TV119 (Ra) > S. enteric SL1181 (Re) > S. marcescens > E. coli F583 (Rd) (Figure 3(b)). The binding of E. coli O111:B4, E. coli O127:B8, E. coli EH100 (Ra), S. enterica TV119 (Ra), and S. enteric SL1181 (Re) was higher in HBS-EP than in HBS-P buffer. Whereas, in the case of K. pneumonia, Pseudomonas aeruginosa, S. enterica, and S. marcescens, the presence of EDTA did not markedly affect the AGP binding levels. Because of its insolubility in the flow buffer, the binding of diphosphoryl lipid A from E. coli was investigated using hybrid SUVs. There was no specific AGP binding detected for diphosphoryl lipid A (data not shown). Hybrid SUV experiments were also performed with fully synthetic Kdo2-lipid A and E. coli F583 (Rd) LPS (Figure 3(d)). There was no specific AGP binding detected for E. coli F583 (Rd) lipid A, while as the concentrations of AGP were higher than 7 μM, a dose-dependent response to Kdo2-lipid A was observed (Figure 3(d)).

Bottom Line:
The ability of AGP to bind circulating lipopolysaccharide (LPS) in plasma is believed to help reduce the proinflammatory effect of bacterial lipid A molecules.In order to dissect the contribution of the lipid A, core oligosaccharide and O-antigen polysaccharide components of LPS, the AGP binding affinity of LPS from smooth strains, were compared to lipid A, Kdo2-lipid A, R(a), R(d), and R(e) rough LPS mutants.The SAR analysis enabled by the binding data suggested that, in addition to the important role played by the lipid A and core components of LPS, it is predominately the unique species- and strain-specific carbohydrate structure of the O-antigen polysaccharide that largely determines the binding affinity for AGP.

ABSTRACTThe ability of AGP to bind circulating lipopolysaccharide (LPS) in plasma is believed to help reduce the proinflammatory effect of bacterial lipid A molecules. Here, for the first time we have characterized human AGP binding characteristics of the LPS from a number of pathogenic Gram-negative bacteria: Escherichia coli, Salmonella typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa, and Serratia marcescens. The binding affinity and structure activity relationships (SAR) of the AGP-LPS interactions were characterized by surface plasma resonance (SPR). In order to dissect the contribution of the lipid A, core oligosaccharide and O-antigen polysaccharide components of LPS, the AGP binding affinity of LPS from smooth strains, were compared to lipid A, Kdo2-lipid A, R(a), R(d), and R(e) rough LPS mutants. The SAR analysis enabled by the binding data suggested that, in addition to the important role played by the lipid A and core components of LPS, it is predominately the unique species- and strain-specific carbohydrate structure of the O-antigen polysaccharide that largely determines the binding affinity for AGP. Together, these data are consistent with the role of AGP in the binding and transport of LPS in plasma during acute-phase inflammatory responses to invading Gram-negative bacteria.